Allergenic disease is the sixth major cause to acute and chronic diseases. The disease has a strong genetic component and has a 30-60% chance of inheritability. Allergic rhinitis affects the eyes, nose and sinuses. It causes stuffy or runny nose, ears and throat postnasal, watery or itchy eyes, and bronchial tube irritation, also known as hay fever. Allergic dermatitis affects the skin, causing an itchy rash. It is also known as contact dermatitis. Asthma affects the lungs, causing shortness of breath or wheezing. Food allergies affect the stomach and other internal organs, and may also cause symptoms to the entire body. Urticaria is a condition resulting with hives on the skin. One type of allergic reaction that requires special attention is anaphylaxis, which is sudden, severe, and potentially fatal, with symptoms that can affect various areas of the body. The symptoms usually appear very quickly after exposure to the allergen and can include intense itching all over the body, full-body swelling, respiratory distress, and can even lead to life threatening shock. Approximately 10-15% of Chinese and around 20% of American population are affected by the allergic disease as well as other countries.
As of present, uses in treating the allergic disease the medicine mostly for the control clinical symptom, steroid hormone, the antihistamine medicine, medicine for reduces the hyperemia, and the bronchial tube relaxant; Recently American FDA has authorized in the clinical test anti-IgE monoclonal antibody. These medicines have the varying degree side effect. Research institutes, the universities and the pharmaceutical companies are seeking a new anti-allergic medicine.
The allergic reaction mechanism is: an immune system reaction to a typically harmless substance. The immune system is always working to fight off parasites, fungi, viruses and bacteria. However, sometimes the immune system will treat a harmless substance (called an allergen) as an unwanted invader and try to fight it. This overreaction of the body's immune system to a typically harmless substance is called an allergic reaction.
A hallmark of the allergic diathesis is the tendency to maintain a persistent IgE response after antigen (allergen) presentation. The initial exposure to antigens stimulates the production of specific IgE molecules, which bind to high-affinity Fc receptors on the surface of mast cells. Upon reexposure of antigens, the cross-linking of antigens and membrane-bound IgE molecules result in the release of vasoactive mediators, setting off subsequent clinical manifestation of sneezing, pruritus, and bronchospasm. Immunoglobulin receptors (also referred to as Fc receptors), are cell-surface receptors of mast cells, that bind to the constant region of immunoglobulins, and mediate various immunoglobulin functions other than antigen binding.
Fc receptors that bind with IgE molecules (a type of immunoglobulin) are found on many types of cells in the immune system. There are two different Fc receptors currently known for IgE, the multichain high-affinity receptor, FcεRI, and the low-affinity receptor, FcεRII. IgE molecules mediate its biological responses as an antibody through these Fc receptors. The high-affinity FcεRI receptor, expressed on the surface of mast cells, basophils, and Langerhans cells, belongs to the immunoglobulin gene superfamily, and has a tetrametric structure composed of an α-chain, a β-chain and two disulfide-linked γ-chains that are required for receptor expression and signal transduction. The α-chains of the receptor interact with the distal portion of the third constant domain of the IgE heavy chain. The specific region of the human IgE molecule involved in binding to the human FcεRI receptor have been identified as including six amino acids, Arg-408, Ser-411, Lys-415, Glu-452, Arg-465, and Met-469. The interaction is highly specific with a binding constant of about 1010M−1.
The low-affinity FcεRII receptor, represented on the surface of inflammatory cells, such as eosinophils, leukocytes, B lymphocytes, and platelets, did not evolve from the immunoglobulin superfamily but has substantial homology with several groups of animals and is made up of a transmembrane chain with an intracytoplasmic NH2 terminus. The low-affinity receptor, FcεRII (CD23), is currently known to have two forms, FcεRIIa and FcεRIIb, both of which have been cloned and sequenced. The two forms differ only in the N-terminal cytoplasmic region, with the extracellular domains being identical. FcεRIIa is normally expressed on B cells, while FcεRIIb is expressed on T cells, B cells, monocytes and eosinophils upon induction by the cytokine IL-4.
Through the high-affinity FcεRI receptor, IgE plays key roles in an array of acute and chronic allergic reactions, including asthma, allergic rhinitis, atopic dermatitis, severe food allergies, chronic urticaria and angioedema, as well as the serious physiological condition of anaphylactic shock. The binding of a multivalent antigen to an antigen-specific IgE molecule, which is specifically bound to a FcεRI receptor on the surface of a mast cell or basophil, stimulates a complex series of signaling events that culminate in the release of host vasoactive and proinflammatory mediators that contributes to both acute and late-phase allergic responses.
The function of the low-affinity FcεRII receptor (also referred to as CD23), found on the surface of B lymphocytes, is less well-established than that of the FcεRI receptor. FcεRII, in a polymeric state, binds to IgE molecules, and this binding may play a role in controlling the type (class) of antibody produced by B cells.
Three groups of Fcγ receptors that bind to the constant region of human IgG molecules have so far been identified on cell surfaces. They are, FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), all of which belong to the immunoglobulin gene superfamily. The three Fcγ receptors have a large number of various isoforms.
In addition to the stimulatory FcεRI receptor, mast cells and basophils also co-express an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing inhibitory low-affinity receptor, called the FcγRIIb receptor, which act to negatively regulate antibody functions. The FcγRIIb receptor belongs in the inhibitory receptor superfamily (IRS), which is a growing family of structurally and functionally similar inhibitory receptors that negatively regulate immunoreceptor tyrosine-based activation motif (ITAM)-containing immune receptors and a diverse array of other cellular responses. Coaggregation of an IRS member (such as FcγRIIb receptor) with an activating receptor (such as FcεRI receptor) leads to phosphorylation of the characteristic ITIM tyrosine and subsequent recruitment of the SH2 domain-containing protein tyrosine phosphatases SHP-1 and SHP-2, and the SH2 domain-containing phospholipases, SHIP and SHIP2. Possible outcomes of the coaggregation include inhibition of cellular activation, as demonstrated by the coaggregation of FcγRIIb and B-cell receptors, T-cell receptors, and activating receptors, such as FcεRI and cytokine. A key contributor to asthma, allergic rhinitis and severe food reactions is the induced IgE-driven mediators released from mast cells and basophils. The cross-linking of a mast cell or basophil FcεRI receptor with a multivalent antigen, activates tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the β- and γ-FcεRI subunit cytoplasmic tails, thereby initiating downstream signaling through Syk. Mast cells and basophils also express the FcγRIIb receptor, which contains a single conserved immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic tail. Studies indicate that the aggregating of FcγRIIb to FcεRI leads to rapid tyrosine phosphorylation of the FcγRIIb ITIM tyrosine by FcεRI-associated Lyn and inhibition of FcεRI signaling. This hypothesis has been supported in experiments using human Ig Fcγ-Fcε fusion proteins that directly cross-link the FcεRI and FcγRIIb receptors on human basophils.
Invention Publication
The goal of this invention is to provide a genetic gene and its encoding fusion protein; this fusion protein has an anti-allergic activity.
The fusion protein used in this invention, named FP4, which is a function protein sequenced amino acid as SEQ ID No 2, or any derivation protein from sequencing of SEQ ID 2.; or any substitution, by decreasing or increasing several amino acid of SEQ ID No 2 amino acid sequence. The FP4 fusion protein from amino acid sequence of SEQ ID 2 contains 554 amino acids (see FIG. 1). FP4 structure is composed of three parts: A area (Fcε) (1-300th amino acids from N-end), B area (Fcγ) (318-554th amino acids from N-end) and C area (hinge) (301-317th amino acids from N-end). Among them, the A area originates from human immunoglobulin IgE and is the ligand of IgE receptor FcεRI; the B area originates from human immunoglobulin IgG and has the ligand of IgG receptor FcγRII; the C area originates from human immunoglobulin IgG, a twisting structure.
The fusion protein, FP4, has the following sequences:                1.) Figure of SEQ ID No. 1: DNA sequence;        2.) Figure of SEQ ID No. 2: Amino acid sequences.        3.) Any DNA sequence has more than 95% homology, and also encodes the same function protein sequence.        
Sequence 1's DNA sequence is composed of 1665 base pair; the reading frame is from the 1st base pair to the 1665th base pair starting with the 5′ end.
Anything including the genes and expression cell lines is under the patent protection of this invention. The primers used to amplify the encoding genes are also included in protection of this invention.
Expansion in any fragment of FP4 is also covered in the extent of this patent's protection.
The second goal of this invention is to introduce a method of expression for fusion protein FP4.
The method of FP4 expression introduced in this invention is to transfect the FP4 gene into SP 2/0 cell in order to get the positive clones which express the fusion protein FP4.
The expression vector is constructed by using regular methods to clone FP4 gene into pSecTag vector.
The third goal of this invention is to present an anti-allergic medicine whose active ingredient is fusion protein FP4.
If necessary, one can add one or multiple carriers to the medicine mentioned above. These carriers include pharmaceutical convention diluents, the excipient, the auxiliaries, the bond, the wetting agent, the disintegration medicinal preparation, the absorption promoter, the active surface agent, the absorption carrier, and the lubricant. When needed, one may also add flavoring agents and sweetening agents among other items.
Medicine from this invention can be used with an intravenous injection, a hypodermic injection, applied to the skin directly among other methods. It also can be used as a nasal spray and as a throat, mouth, skin, or membrane inhaler. In addition, this medicine can be applied as a nasal aqueous suspension, eye drops, or ear drops. Furthermore, it can be used as a rectal gel, pill, powder, ball pill, capsule, solution, oil cream, cream, and various other forms. The methods mentioned above can be pharmaceutically prepared with conventional methods to determine the correct dosages.
All the medicines mentioned above generally have a dosage of 0.01-lug/kg/day and the treatment course generally lasts 10 to 20 days.